The paper in Nature Communications is here: https://go.nature.com/2IJNuzn
Cytosolic protein delivery is of great importance for biotherapeutics and fundamental biological investigations, but is challenging due to the lack of efficient and safe transduction technologies. Conventional approaches for cytosolic protein delivery include: (1) physical techniques (i.e. electroporation); (2) fusion of proteins with cell penetrating ligands; and (3) using protein transduction vehicles such as liposomes, polymers or nanoparticles. Although established, those approaches still possess some limitations such as low transduction efficacy, the need of protein modification, or complicated synthesis.
In 2014, my research group at East China Normal University reported the dramatic efficacy of fluorinated polymers in gene delivery (Nat. Commun., 2014, 5, 3053). The polymers were observed to achieve promising performance at extremely low polymer doses, and thus ensured low cytotoxicity (Angew. Chem. Int. Ed., 2015, 54, 11647). The fluoroalkane functionalities on polymers are both hydrophobic and lipophobic, and therefore are beneficial for addressing the multiple barriers during gene delivery such as serum tolerance, cellular uptake, endosomal escape, and intracellular payload release. Considering the similarity of protein delivery and gene delivery, especially the intracellular barriers existing in both delivery processes, we hypothesized to use fluorinated polymers for intracellular protein transduction. However, the road to the development of fluorinated polymers for this purpose is not smooth. Nucleic acids bind to fluorinated polymers via electrostatic interactions in gene delivery, but the proteins have limited charges and the charge state is uncertain at physiological conditions, and thus the experiences got on gene delivery are not applicable for cytosolic protein delivery. It is not surprising that the efficient fluorinated polymers discovered in gene delivery do not work at all for protein delivery. Therefore, how to design fluorinated polymers that can efficiently bind proteins becomes the major challenge.
Fluorinated polymers usually possess excellent self-assembly property in aqueous solution, and their aggregated structures depend on the fluoroalkane chain length and density. We therefore speculated that these parameters are critical for polymer assembly and protein encapsulation. In this case, Miss Zhenjing Zhang, a master student in my group, developed a library of fluorinated polymers with various chain lengths and grafting density to screen efficient protein carriers. Fortunately, she finally got two promising materials in the screening pool and successfully revealed the advantage of fluorinated polymers over other non-fluorinated polymers for intracellular protein delivery (Figure 1). The fluorous substituents on the polymer play essential roles in the formation of uniform nanoparticles with proteins, avoiding protein denaturation, efficient cell internalization, and minimizing cytotoxicity. As expected, a balance on fluorophilicity is critical for efficient protein transduction. Excess fluorophilicity on the polymer may lead to the pre-assembly of fluorinated polymers into stable vesicles, which hinders efficient protein encapsulation. This should be avoided when designing fluorinated polymers for cytosolic protein delivery.